The present invention relates to novel chelating agents and the manganese chelated complex salts thereof, the physiologically compatible salts thereof and the use of these compounds in magnetic resonance imaging (MRI).
A valuable M.R.I. contrast agent should recognizedly have, in addition to low administration dosages, very good relaxivity, so as to provide a suitable increase in the contrast between lesions and healthy tissue and among the different organs and tissues; high thermodynamic stability; slow transmetallation kinetic, in particular with ions of endogenous metals such as magnesium and calcium; low toxicity and very good tolerability. The gadolinium chelated complexes are at present the preferred contrast agents, due to the properties of Gd3+ ion which has seven discoupled electrons and the highest magnetic moment. At the present time, commercially available contrast agents containing gadolinium chelated complexes are: Magnevist(R) (Gd-DTPA meglumine double salt), Dotarem(R) (Gd-DOTA meglumine salt), Omniscan(R) (Gd-DTPA-BMA) and ProHance(R) (Gd-HP-DO3A).
The more recent searches aim at founding contrast agents which, besides having the above cited characteristics, are also specific for a definite tissue or body region. Manganese has been suggested as an alternative to gadolinium in these tissue-specific contrast agents (Investigative Radiology 1995, 30(10), 611-620).
The potential usefulness of the Mn2+ ion as an M.R.I. contrast agent has been taken in consideration since 1978 for myocardium imaging. Manganese complexes with porphyrines and derivatives thereof (for example Mn(III)-meso-tetra-(4-sulfonatophenyl)porphyrine, Mn-TPPS4) were proposed as contrast agents specific for tumors (Investigative Radiology, cited ref.). In Magnetic Resonance in Medicine 1988, 8, 293-313, the longitudinal proton relaxation times in rabbit tissues have been studied after intravenous administration of MnCl2 and of Mn-PDTA (1,3-propylenediamino-N,Nxe2x80x2,Nxe2x80x3,Nxe2x80x2xe2x80x3-tetraacetate). In a recent study, M.R.I. contrast agents were evaluated consisting of Mn-EDTA lipohilic derivatives (for example, manganese-EDTA-bis(hydroxypropyldecylamine), Mn-EDTA-DDP) bound to the membranes of small unilamellar liposomes (xe2x80x9cmemsomesxe2x80x9d) , which are potentially valuable for hepatic and cardiac perfusion imaging (Journal of Liposome Research 1994, 4(2), 811-834). The same compounds are object of International Patent Application WO 92/21017 and of U.S. Pat. No. 5,312,617, which disclose M.R.I. contrast agents based on manganese chelates (II) consisting of EDTA bis-amides where the amide nitrogens are substituted with long chain alkyl residues (C7-C30). These chelates per se or, more preferably, in combination with lipids or liposomes, are reportedly particularly useful for imaging of liver and as blood-pool agents.
M.R.I. contrast agents comprising manganese chelated complexes are also described in U.S. Pat. Nos. 4,980,148 and 5,246,696. Said complexes, in which the ligand is an alkylenediaminotetraacetic acid in which the alkylene chain is interrupted by one or more substituents selected from O, S, CHOH, CHSH, are stated to be particularly useful for imaging of liver, kidneys, pancreas and gastrointestinal tract.
Recently, Teslascan(R) (manganese complex of N,Nxe2x80x2-1,2-ethanediylbis[N-[[3-hydroxy-2-methyl-5-[(phosphono-oxy)methyl]-4-pyridinyl]-methyl]glycine salified with Na+ (1:3), mangafodipir trisodium, was marketed in Europe and in the U.S.A. for use in M.R.I. of the liver. This compound was considered useful for M.R.I. diagnosis of pancreas adenocarcinoma and pancreatitis (Investigative Radiology 1995, 30(10), 611-620).
Ions of paramagnetic metals are known to be highly toxic. The same applies for Mn2+ ion at the doses commonly used for diagnostic imaging, although this is an essential oligoelement, present in all mammal cells.
It is therefore important also in the case of manganese, for this to be administered as stable complex, thereby preventing any toxicity due to the free metal. Conversely, some compounds of the above cited literature, for example Mn-DPDP, show some instability in vivo: a recent biodistribution study proved that this complex dissociates, releasing manganese which accumulates in liver, pancreas and kidneys, whereas the undissociated chelate is removed through glomerular filtration. The M.R.I. properties of Mn-DPDP would therefore be ascribable mainly to the manganese ion released by the complex, which accumulates in liver and pancreas (Investigative Radiology 1994, 29(2), S249-S250). Another recent study gave evidence of the dissociation of Mn-DPDP in liver homogenate, in the presence of calcium and magnesium ions (MRM 1996, 35, 14-19).
The present invention relates to stable manganese chelated complex salts in which said ion is in the oxidation state +2 (Mn(II)). Said compounds are ethylenediaminotetraacetic acid (EDTA) derivatives, characterized in having a substituent containing at least one cyclic unit in xcex1 position to the carboxyl of one or two of the four acetic groups. Contrary to the manganese chelated complexes of the prior art, which, as mentioned above, are generally considered particularly useful in imaging of liver, pancreas and gastrointestinal tract, the chelates of the present invention have a good stability as well as surprising relaxivity values in human serum.
More particularly, the invention relates to compounds of formula (I), both in the racemic and optically active forms: 
wherein:
R1, R2 are independently a hydrogen atom, or a straight or branched (C1-C20) alkyl chain, saturated or unsaturated, being said chain optionally interrupted by one or, more nitrogen or sulfur atoms, as well as by xe2x80x94COxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94NHCOxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94SO2NHxe2x80x94 groups, or optionally substituted by one or more NH2, OH, halogen, COOH groups and the respective ester or amide derivatives, said chain being in any case interrupted or substituted by one or more R3 cyclic residues, which are the same or different, non-fused or fused, with the proviso that, when some of the R3 residues are fused together, the maximum number of rings forming the corresponding polycyclic unit is three, wherein
R3 is a 5- or 6-membered cyclic unit, carbocyclic or heterocyclic, saturated, unsaturated or aromatic, being said cyclic units unsubstituted or substituted with one or more R4 groups, which are the same or different, wherein
R4 is OH, halogen, NHR5, N(R5)2, xe2x80x94Oxe2x80x94R5, xe2x80x94Sxe2x80x94R5, or xe2x80x94COxe2x80x94R51 wherein the R5 groups, which are the same or different, are a straight or branched (C1-C5) alkyl, unsubstituted or substituted with one or more hydroxy and/or alkoxy and/or carboxy groups, or R4 is a COOH group, or an ester or amido derivative thereof, or a xe2x80x94SO3H group or an amido derivative thereof,
or R4 is a xe2x80x94Oxe2x80x94R6 group, wherein R6 is a 5- or 6-membered cyclic unit, carbocyclic or heterocyclic, saturated, unsaturated or aromatic, being said cyclic unit optionally substituted by one or more OH, halogen, xe2x80x94NHR5, xe2x80x94N(R5)2, xe2x80x94Oxe2x80x94R5, xe2x80x94Sxe2x80x94R5, xe2x80x94COxe2x80x94R5 groups, wherein R5 has the meanings defined above, or by one or more xe2x80x94COOH groups, or the ester or amido derivatives thereof, or xe2x80x94SO3H or amido derivatives thereof,
with the proviso that R1 and R2 are not at the same time hydrogen;
as well as the chelates thereof with the manganese ion in the oxidation state +2 (Mn(II)) and the salts thereof with physiologically compatible organic bases selected from primary, secondary, tertiary amines or basic amino acids, or with inorganic bases whose cations are sodium, potassium, magnesium, calcium, or mixtures thereof.
In case the chelated complex has a total charge, this is preferably neutralized with a physiologically compatible counter-ion. Suitable substances for salifying the compounds of the invention and/or the chelates thereof, are, for example:
cations of inorganic bases such as alkali or alkaline-earth metals ions selected from sodium, potassium, magnesium, calcium, or mixtures thereof;
cations of physiologically compatible organic bases selected from primary, secondary and tertiary amines such as ethanolamine, diethanolamine, morpholine, glucamine, N-methylglucamine, N,N-dimethylglucamine;
cations of amino acids such as those of lysine, arginine or ornithine.
Particularly preferred are N-methylglucamine salts.
In the compounds of formula (I), particularly preferred meanings for R1 and R2 are the following: 
Particularly preferred classes of compounds within formula (I), are those of formula (II): 
and of formula (III): 
wherein:
R7 is a 5- or 6-membered cyclic unit, carbocyclic or heterocyclic, saturated, unsaturated or aromatic, optionally substituted by one or more groups selected from OH, halogen, COOH or an ester or amido derivative thereof, xe2x80x94SO3H or an amido derivative thereof, or xe2x80x94R5, xe2x80x94NHR5, xe2x80x94N(R5)2, xe2x80x94Oxe2x80x94R5, xe2x80x94Sxe2x80x94R5, wherein R5 has the meanings defined above, or from a group selected from xe2x80x94Oxe2x80x94R8 and xe2x80x94CH2R8 wherein R8 is a further 5- or 6-membered cyclic unit, carbocyclic or heterocyclic, saturated, unsaturated or aromatic, optionally substituted by one or more groups selected from OH, COOH and halogen;
n=1-6.
Particularly preferred classes of compounds within formula (I), are also those of formula (IV) 
and of formula (V), respectively, 
in which:
R9 is a group comprising 2 or 3 cyclic units, nonfused or fused, which are the same or different, wherein said units can be carbocyclic, heterocyclic, saturated, unsaturated or aromatic;
n=1-6;
m=0 or 1;
X=xe2x80x94NHCO, xe2x80x94CONH, xe2x80x94CONHxe2x80x94CH2xe2x80x94.
Furthermore, within formulae (II) and (III), particularly preferred compounds are those in which R7 is cyclohexyl, phenyl, hydroxyphenyl or a 3,5-diiodothyronine residue; within formulae (IV) and (V), particularly preferred compounds are those in which R9 is naphthalene, anthracene or indole and X is xe2x80x94NHCO.
Among the possible manganese chelates included within formula (I), most preferred are those having as ligands the compounds shown hereinbelow: 
The present invention also relates to the use of the Mn2+ chelated complexes of the chelating compounds of formulae (I) to (V) and to the salts thereof, for the preparation of pharmaceutical compositions for diagnostic use, as well as the formulations themselves.
The chelated complexes of the invention have, in addition to a good stability, surprising relaxivity values in human serum. These characteristics allow to foresee interesting applicative uses thereof, in view of an improvement in the images obtainable using said compounds in the preparation of M.R.I. contrast agents for general use, as well as the possible use thereof in specific formulations for the imaging of the cardiocirculatory system.
More particularly, the chelates of the present invention are characterized in having particularly high r2 values in human serum. This makes them most suitable, and this is a further aspect of the present invention, for use in M.R.I. diagnostic imaging in recording images of organs or tissues, wherein said images are acquired by T2 weighed sequences. Furthermore, the considerable increase in relaxivity in human serum shown by the compounds of the invention allows the use thereof in diagnostic formulations requiring a low dosage of the contrast agent, said formulations providing good quality images, while assuring a significant improvement from the toxicologic point of view.
The contrastographic formulations containing the chelates of the invention can therefore be formulated with concentrations of contrast agent ranging from 0.001 to 1.0 mmol/mL, preferably from 0.01 to 0.5 mmol/mL; in particular, concentrations lower than 0.25 mmol/mL for the low-dosage formulations.
Said formulations can be administered to the patient in doses ranging from 0.001 to 0.1 mmol/kg, preferably 0.1 mmol/kg; in particular, doses ranging from 5 to 50 xcexcmol/kg when administered in low dosages.
According to the present invention, said chelated complexes proved to be particularly suitable also in the preparation of pharmaceutical formulations for M.R.I. diagnostic imaging at low fields, i.e. fields ranging from 0.1 to 0.5 Tesla or, when measuring the intensity of the applied magnetic field in Hertz, fields below 20 MHz (An introduction to Magnetic Resonance in Medicine, Ed. Peter A. Rinck). The compounds of the invention, thanks to their excellent relaxivity shown even at such low fields, can advantageously be used with apparatuses such as Artroscan (arthrographs which make use of the Magnetic resonance), open apparatuses, apparatuses intended for diagnostic of single regions of the human body (e.g. the shoulder), and generally apparatuses less expensive, easier to handle and increasingly diffused.
The chelates of the invention have also shown a good applicability in the imaging of the liver and of the biliary tracts, which organs are reached by said chelates, thus confirming their characteristics of stability.
Among the possible synthetic routes which can be used for the preparation of the compounds of the invention, those illustrated in the general schemes reported in the following are preferred.
When only one of R1 or R2 is different from hydrogen, a possible process is illustrated in the following Scheme 1: 
wherein:
R1 has the same meanings as in formula (I);
X=halogen (Cl, Br, I);
m=number of the total charges of the chelated complex;
B=substance suitable for salifying the chelated complex (for example Na+, K+, Mg++, Ca++ or mixtures thereof, meglumine, etc.);
z=number of the charges of B;
p is an integer such that the product: pxc2x7z=m.
In step (a1) an xcex1-amino acid (1A), natural or synthetic, is reacted with a suitable halide (e.g. KBr) in acid aqueous medium, in the presence of NaNO2 and at a temperature which can range from xe2x88x925 to +5xc2x0 C. The resulting xcex1-haloacid (2A) is reacted, in step (b1), with 1,2-diaminoethane in aqueous solution, at a temperature from 20 to 40xc2x0 C. The resulting intermediate (3A) is carboxymethylated in step (c1), in basic medium at pH 10 with an xcex1-haloacetic acid (e.g. bromoacetic acid) at about 60xc2x0 C., to give the free ligand (4A). The latter is reacted in step (d1) with the stoichiometric amount of manganese, as salt, in the presence of the base amount necessary for the neutralization; the reaction is preferably carried out in water or in a suitable water-alcohol mixture, at 25 to 40xc2x0 C.; thereby obtaining the desired chelated complex (5A).
When both R1 and R2 are different.from hydrogen and R1 is the same as R2, the procedure summarized in the following Scheme 2 will be followed: 
wherein:
R1 has the same meanings as in formula (I);
X=halogen (Cl, Br, I);
m=number of the total charges of the chelated complex;
B=substance suitable for salifying the chelated complex (for example Na+, K+, Mg++, Ca++ or mixtures thereof, meglumine, etc.);
z=number of the charges of B;
p is such an integer that the product: pxc2x7z=m.
In step (a2), a suitable xcex1-amino acid (1B) is reacted with a 1,2-dihaloethane, such as 1,2-dibromoethane (2B) in a suitable solvent (e.g. water/ethanol, water/methanol mixtures), keeping pH at about 9 with a suitable buffer (preferably 2M borate buffer) at a temperature which can range from 60 to 90xc2x0 C. Alternatively, (step axe2x80x22), an xcex1-haloacid (1Bxe2x80x2) can be reacted with 1,2-diaminoethane (2Bxe2x80x2) in aqueous solution at 20 to 60xc2x0 C. The resulting intermediate (3B) is carboxymethylated in step (b2) with an xcex1-haloacetic acid (e.g. bromoacetic acid) at basic pH (about 10) and at a temperature from 40 to 70xc2x0 C., to give the free ligand (4B) . This is reacted in step (c2) with the stoichiometric amount of manganese, according to the general procedure already described for scheme 1, to obtain the desired chelated complex (5B).
Using a different stoichiometry, asymmetric ligands can be prepared, wherein R1 and R2 are both different from hydrogen and different from each other, as represented in the following Scheme 3: 
wherein:
R1, R2 have the same meanings as in formula (I);
X=halogen (Cl, Br, I);
m=number of the total charges of the chelated complex;
B=substance suitable for salifying the chelated complex (for example Na+, K+, Mg++, Ca++ or mixtures thereof, meglumine, etc.);
z=number of the charges of B;
p is an integer such that the product: pxc2x7z.=m.
An alternative process which provides disubstituted compounds is represented by the following Schemes 4A and 4B: 
wherein:
R1 has the same meanings as in formula (I);
Pg=protective group (e.g. t-butyl)
Y=Cl, Br or other leaving groups (I, xe2x80x94OMs, xe2x80x94OTf, xe2x80x94OTs);
Step (d1) comprises the protection of the alcoholic fraction of a 2-haloethanol (preferably 2-bromoethanol) with dihydropyran, to give intermediate (2D). The alcohol-protecting group can be, for example, benzyl or trityl. The reaction takes place in an organic solvent such as CH2Cl2, CHCl3, CH2ClCH2Cl, in the presence of 4-toluenesulfonic acid pyridinium salt or of another acid catalyst. In intermediate (2D), the leaving group is preferably Br.
In step (d2) the ester (e.g. t-butyl ester) of a natural or synthetic xcex1-amino acid (1D), either in the racemic or optically active form, is reacted with the intermediate (2D) in the presence of a base, such as diisopropylethylamine, in a solvent such as CH3CN, DMF or a chlorinated solvent, to give intermediate (3D).
The latter is reacted, in step (d3), with .a bromoacetic acid ester (e.g. t-butyl-bromoacetate) in the presence of a base, such as diisopropylethylamine, to give intermediate (4D) which is reacted, in the subsequent step (d4), with 4-toluenesulfonic acid pyridinium salt or another acid catalyst, in an ethanol/water mixture, at a temperature of 20-60xc2x0 C., to give intermediate (5D).
In step (d5), intermediate (5D) is brominated with N-bromosuccinimide in the presence of triphenylphosphine, to give intermediate (6D), which is reacted, in step (d6), with the ester (e.g. t-butyl ester) of an xcex1-amino acid (7D), in a suitable solvent (e.g. CH3CN/buffer phosphate pH 8), to give intermediate (8D). This is then reacted with a bromoacetic acid ester (e.g. t-butyl-bromoacetate) in the presence of a base, such as diisopropylethylamine, to give tetraester (9D). The latter is deprotected in step (d8) with known methods (e.g. hydrolysis with CF3COOH or (CH3)3SiI, when the protective group is t-butyl) to give the free ligand (10D), which is subsequently complexed and salified according to the general procedure already described in the above schemes. 
wherein:
R1, R2 have the same meanings as in formula (I);
Pg=protective group (e.g. t-butyl);
X=halogen (Cl, Br, I).
The following examples illustrate the best experimental conditions to prepare the compounds of the invention.